Medicinal uses of mu-opioid receptor agonists

ABSTRACT

The present invention provides methods for stimulating a mu-opioid receptor agonist peptide in a mammal in need thereof. The methods comprise administering to the mammal an effective amount of a selective mu-opioid receptor agonist peptide that comprises at least two α-amino acid residues. At least one of the amino acid residues has a positive charge. The amino acid residue in the first position is a tyrosine or tyrosine derivative. The amino acid in the second position is a D-α-amino acid. The present invention also provides methods of treating a mammal suffering from conditions or diseases by administering to the mammal an effective amount of the peptides.

This application claims priority from U.S. provisional application Ser.No. 60/219,046 filed Jul. 18, 2000, the contents of which areincorporated herein by reference.

This invention was made with Government support from the NationalInstitute on Drug Abuse under Grant No. P01 DA08924. The Government hascertain rights in this invention.

The invention as claimed herein was made pursuant to a joint researchagreement, within the meaning of 35 U.S.C. § 103(c) between CornellResearch Foundation, Inc. and Institut de Recherches Cliniques deMontreal. The joint research agreement was in effect on or before thedate the claimed invention was made, and the claimed invention was madeas a result of activities undertaken within the scope of the jointresearch agreement.

BACKGROUND OF THE INVENTION

Opiates (derived from the opium poppy) or opiate-like (synthetic) drugsare widely used to alleviate moderate to severe pain. These drugs areclassified together as opioids. Opioids derived from the opium poppyinclude morphine and codeine. Opiate-like or synthetic drugs includefentanyl, meperidine and methadone.

Opioids bind to specific receptor molecules. Distinct categories ofopioid receptors have been identified which include mu, delta and kappareceptors. (W. Martin et al., J. Pharmacol. Exp. Ther. 197, 517 (1977)).

Opioids are useful for various kinds of pain management. In particular,opioids are used to alleviate postoperative pain and chronic pain, suchas cancer and neuropathic pain, and pain during labor and delivery.However, opioid use has been linked to many dangerous side effects, suchas tolerance or physical dependence, constipation, cardiac depressionand respiratory depression.

One major concern with opioid use has been the transfer of opioidsacross the placenta to the fetus. Opioids may adversely affect the fetusby compromising the delivery of oxygen and substrates from the mother.The respiratory depressant effects of opioids may also decrease fetaloxygen availability.

Another concern with the currently available opioids is that they aretoo short-acting for labor pain. For example, fentanyl usually providesrelief for only 60 to 90 minutes. Labor pain can last up to twelvehours.

In addition, due to the depression of cardiac and respiratory functionseen with opioids, dangers exist when opioids are used before and duringsurgery when the patient has compromised cardio-respiratory functions.For example, respiratory depression is especially risky for individualswho have compromised respiratory systems, such as asthmatics and smokers

Opioids negatively affect motor function and are also associated withundesirable sedative effects. This is problematic for orthopedic andjoint replacement surgeries, which require immediate post-operativemotility.

Accordingly, there is a need for pain management treatments that do notdepress cardiac or respiratory function and do not affect motorfunction.

Further, there is a need for new pain management treatments that do notcross the placental barrier or compromise the maternal respiratory andcardiac function. In addition, longer acting treatments for pain areneeded.

Additionally, it would be beneficial to have a pain management treatmentthat will be effective in mammals that have developed a tolerance toopioids.

SUMMARY OF THE INVENTION

The present invention is directed to a method for stimulating amu-opioid receptor in a mammal in need thereof. The method comprisesadministering systemically to the mammal an effective amount of aselective mu-opioid receptor agonist peptide. The peptide comprises atleast two α-amino acid residues, at least one α-amino acid residuehaving a positive charge. The α-amino acid residue in the first positionis tyrosine or a tyrosine derivative, and the α-amino acid residue inthe second position is a D-α-amino acid.

In an additional embodiment, the present invention is directed to amethod for stimulating a mu-opioid receptor in a mammal in need thereof.The method comprises administering intrathecally or orally to the mammalan effective amount of a selective mu-opioid receptor agonist peptideselected from the group consisting of2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Tyr-D-Ala-Phe-Phe,2′,6′-dimethyl-Tyr-Ala-Phe-Phe-NH₂ and2′,6′-dimethyl-Tyr-D-Arg-Phe-Orn-NH₂.

Another embodiment of the invention is directed to a method for reducingpain in a mammal in need thereof without risk of respiratory depression.The method comprises administering to the mammal an effective amount ofa selective mu-opioid receptor agonist peptide selected from the groupconsisting of 2′, 6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Tyr-D-Ala-Phe-Phe,2′, 6′-dimethyl-Tyr-D-Ala-Phe-Phe-NH₂ and 2′,6′-dimethyl-Tyr-D-Arg-Phe-Orn-NH₂.

Another embodiment of the invention is a method for protecting amammal's heart from ischemic-reperfusion injury before, during and/orafter cardiac surgery. The method comprises administering to the mammalan effective amount of a selective mu-opioid receptor agonist peptide.The peptide comprises at least two α-amino acid residues with at leastone α-amino acid residue having a positive charge. The α-amino acidresidue in the first position is a tyrosine or tyrosine derivative andthe α-amino acid residue in the second position is a D-α-amino acid.

In yet another embodiment of the invention, is a method for inhibitingnorepinephrine uptake in a mammal in need thereof. The method comprisesadministering an effective amount of a selective mu-opioid receptoragonist peptide. The peptide comprises at least two α-amino acidresidues with at least one α-amino acid residue having a positivecharge. The α-amino acid residue in the first position is a tyrosine ortyrosine derivative and the α-amino acid residue in the second positionis a D-α-amino acid.

In a final embodiment of the invention, is a method for treating cardiacfailure or angina in a mammal in need thereof. The method comprisesadministering an effective amount of a selective mu-opioid receptoragonist peptide. The peptide comprises at least two α-amino acidresidues with at least one α-amino acid residue having a positivecharge. The α-amino acid residue in the first position is a tyrosine ortyrosine derivative and the α-amino acid residue in the second positionis a D-α-amino acid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Dose-dependent antinociceptive effects of intrathecal MOR,DALDA, and [Dmt¹]DALDA in the tail-flick test (n=10 in each group).

FIG. 2. Time course of antinociceptive effects of intrathecal MOR,DALDA, and [Dmt¹]DALDA. A dose of 3 times the ED₅₀ value was given.Tail-flick latencies were measured before and every hour up to 15 hafter the administration of the drug (n=8 in each group). *Significantlydifferent from baseline values (p<0.05).

FIG. 3. Effects of intrathecal MOR, DALDA, and [Dmt¹]DALDA on minuteventilation during hypercapnic breathing. Doses of 3 times the ED₅₀value and 30 times the ED₅₀ value (antinociceptive effect) of each drugwere administered. The smallest minute ventilation value (minimum minuteventilation) during the postadministration period was determined andexpressed as percentage of baseline minute ventilation. n=6-10 in eachgroup. *Significantly different from saline group (p<0.05).

FIG. 4. Effect of yohimbine on the antinociceptive effects of[Dmtl¹]DALDA. [Dmt¹]DALDA (1.1 pmol) alone, [Dmt¹]DALDA (1.1 pmol) andyohimbine (100 μg), or yohimbine (100 μg) alone were given intrathecallyto rats. Tail-flick latencies were measured before and every 20 minafter drug administration. Yohimbine significantly attenuated theantinociceptive effect of [Dmt¹]DALDA (p<0.05, two-way ANOVA) while nothaving any effect on tail-flick latency by itself.

FIG. 5. Effect of yohimbine on the antinociceptive effects of DALDA.DALDA (240 pmol) alone, DALDA (240 pmol) and yohimbine (100 μg), oryohimbine (100 μg) alone were given intrathecally to rats. Tail-flicklatencies were measured before and every 20 min after drugadministration. The antinociceptive effect of DALDA was notsignificantly attenuated by yohimbine.

FIG. 6. Effects of MOR, DALDA, and [Dmt¹]DALDA on NE uptake is spinalcord synaptosomes. Each value represents the mean±S.E. determined fromthree to five experiments. The IC₅₀ values for [Dmt¹]DALDA, DALDA, andMOR were 4, 54, and 870 μg, respectively.

DETAILED DESCRIPTION

It has now been discovered that certain selective mu-opioid receptoragonist peptides can be used for pain management. The peptides havefewer of the dangerous side effects associated with conventionalopioids.

The peptide comprises at least two α-amino acid residues. In thisspecification, an α-amino acid residues may be any naturally occurringor non-naturally occurring D or L α-amino acid. The naturally occurringamino acids are typically the twenty most common amino acids, i.e.alanine (ala), arginine (arg), asparagine (asn), aspartic acid (asp),cysteine (cys), glutamine (glu), glutamic acid (glu), glycine (gly),histidine (his), isoleucine (ileu), leucine (leu), lysine (lys),methioine (met), phenylalanine (phe), proline (pro), serine (ser),threonine (thr), tryptophan, Trp), tyrosine (tyr), and valine (val).

The non-naturally occurring amino acids can be any organic moleculewhich contains an amino acid group. For example, the non-naturallyoccurring amino acids may be a derivative of a naturally occurring aminoacid. Some examples of non-naturally occurring α-amino acids includehomolysine, 2,3 or 2,4-diaminobutyric acid, 2,3-diaminopropionic acid,ornithine, norleucine, and norvaline.

The residue at first position of the peptide, i.e. the N-terminalposition, is tyrosine or, preferably, a derivative of tyrosine. Aderivative of tyrosine is a tyrosine with one or more structuralmodifications. Structural modifications of tyrosine include, forexample, the addition of one or more groups to the benzene ring or tothe amino group.

For example, one or more groups can be added to one or more of the 2′,3′, 5′, or 6′ position of the benzene ring. The group can be any groupthat can be added to a benzene ring. Some examples of such groupsinclude hydroxy, C₁-C₄ alkoxy, amino, methylamino, dimethlyamino, nitro,halo (fluoro, chloro, bromo, or iodo), or branched or unbranched C₁-C₄alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, ort-butyl.

Some examples of groups that can be added to the amino group of tyrosineinclude the C₁-C₄ alkyl groups mentioned above. Preferred derivatives oftyrosine include 2′-methyltyrosine (mmt), 2′,6′-dimethyltyrosine (dmt),N,2′,6′-trimethyltyrosine (tmt) and 2′-hydroxy-6′-methyltyrosine (hmt).

The α-amino acid residue in the second position of the peptide is aD-α-amino acid. The D-α-amino acid may be any naturally occurring ornon-naturally occurring D α-amino acid. The remaining amino acidresidues can be any naturally or non-naturally occurring D or L-α-aminoacid residues.

The peptide preferably comprises no more than seven amino acid residues.More preferably, the peptide comprises no more than five amino acidresidues. Most preferably, the peptide comprises four amino acidresidues.

The peptide may be a linear peptide. Alternatively, the peptide is acyclic peptide. In a cyclic peptide, a first group, preferably acarboxyl group, forms a covalent bond with a second group, preferably anamino group. The first group is preferably on the C-terminal amino acid.The second group is preferably on the N-terminal amino acid.

The mu-opioid receptor peptide of the present invention has at least oneα-amino acid residue that has a positive charge at physiological pH.Preferably at least two or three α-anino acid residues have a positivecharge at physiological pH. Some examples of α-amino acid residues thathave a positive charge at physiological pH include tyrosine and tyrosinederivatives, arginine, lysine, histidine, 2,3- or 2,4-diaminobutyricacid, and 2,3-diaminopropionic acid.

In a preferred embodiment, the C-terminal residue of the peptide is anamide derivative of the carboxylate group. The amide group may be, forexample, amido, N-methylamido, N-ethylamido, N,N-dimethylamido, orN,N-diethylamido.

Some examples of peptides include H-Tyr-D-Arg-Phe-Lys-NH₂ (DALDA) and2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂ ((dmt¹)DALDA). Examples of otherpeptides include Tyr-D-Ala-Phe-Phe (TAPP),2′,6′-dimethyl-Tyr-D-Ala-Phe-Phe-NH₂, and2′,6′-dimnethyl-Tyr-D-Arg-Phe-Orn-NH₂. (An —NH₂ after the C-terminalamino acid residue indicates an amide group.)

In one embodiment, the invention relates to a method for stimulating amu-opioid receptor in a mammal. The method comprises administering tothe mammal an effective amount of any selective mu-opioid receptoragonist peptide described above. In this embodiment, the peptide isadministered systemically. In this specification, systemicadministration means intravenous, subcutaneous, or intramuscularadministration.

In view of prior art teachings to the contrary, see Clapp et al, Am. J.Obstet. Gynecol. 178, 397-401 (199w), it is e that the peptides areeffective when administered systemically. Clapp et al, teach thatmu-opioid selective agonist peptides like those of the present inventionare not expected to produce sufficient analgesia when administeredsystemically.

In another embodiment, the method relates to a method for stimulating amu-opioid receptor in a mammal comprising administering intrathecally ororally to the mammal an effective amount of a selective mu-opioidreceptor agonist peptide. The peptide is selected from the groupconsisting of 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Tyr-D-Ala-Phe-Phe,2′,6′-dimethyl-Tyr-D-Ala-Phe-Phe-NH₂ and2′,6′-dimethyl-Tyr-DArg-Phe-Orn-NH₂.

In another embodiment the invention relates to a method for reducingpain in a mammal without risk of respiratory depression. Respiratorydepression refers to a decrease in the rate and depth of respiration.The method comprises administering to the mammal an effective amount ofa selective mu-opioid receptor agonist peptide. The peptide is selectedfrom the group consisting of 2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂,Tyr-D-Ala-Phe-Phe, 2′,6′-dimnethyl-Tyr-D-Ala-Phe-Phe-NH₂ and2′,6′-dimethyl-Tyr-D -Arg-Phe-Orn-NH₂. The peptide may be administeredby any mode of administration. Systemic, intrathecal, or oraladministration is preferred.

In another advantage, administration of the peptides of the presentinvention is not accompanied by constipation.

A disadvantage of the opioids of the prior art is that they have theability to cross the placental barrier and cause adverse effects in afetus. For example, neonatal respiratory depression and changes in theneurobehavior of a child have been associated with the use of opioidsduring labor and delivery when the opioids are transferred across theplacenta.

One advantage of the peptides of the present invention is their limitedability to cross the placental barrier. Accordingly, the peptides of thepresent invention avoid the undesirable side effects mentioned above.

A major advantage of the methods of the invention is that they areuseful in mammals already tolerant to opioids. Tolerance to opioidsrefers to a decrease in the effects of an opioid at its previous dose,or the need for a higher dose to maintain the same effect.Cross-tolerance is a phenomenon where an individual who has developedtolerance to one opioid, usually is tolerant to other opioids.

The peptides used in the methods of the present invention do not exhibitcross-tolerance. Accordingly, the peptides are effective in anindividual who has developed tolerance to other opioids, but remains inneed of pain reduction. This situation may, for example, occur inindividuals who suffer from chronic pain, such as pain from cancer orneuropathic pain. Neuropathic pain refers to pain that results from adisturbance of function or pathologic change in the nervous system,including the central nervous system and peripheral nervous system.

For these and other reasons, the peptides of the invention are effectiveas analgesic or anesthetic agents. For example, the peptides aresuitable for treating pain caused by surgery. The peptides may beadministered before, during, or after surgery. The method is useful inany type of surgery including cardiac surgery, joint replacement surgeryor transplantation surgery.

In addition to their analgesic properties, the peptides of the presentinvention have been shown to improve myocardial contractile force andcardiac performance. Accordingly, the peptides of the invention areparticularly useful in mammals that suffer from painful conditions, andare also in need of improving myocardial contractile force or cardiacperformance.

For example, the peptides are useful for preconditioning and protectinga heart before and/or during cardiac surgical procedures. Suchprocedures include, for example, coronary bypass and angioplasty. Thepeptides of the present invention can be given prior to or duringcardiac surgery to precondition the heart, to protect it againstischemic damage, and/or to relieve pain.

Moreover, the peptides can be given before, during and/or after cardiacsurgery, such as coronary bypass, to provide both pain relief andprotection against myocardial ischemia-reperfusion injury. After amyocardial ischemic episode, an immediate goal is to reperfuse the heartmuscles, i.e. restoring blood flow to the heart. Early reperfusion,after an ischernic episode, minimizes the extent of heart muscle damageand preserves the pumping function of the heart. By improving myocardialcontractile force, the peptides of the invention can facilitatereperfusion after an ischemic episode.

The method of the present invention is beneficial at any other time amammal is in need of improving myocardial contractile force or cardiacperformance of a heart. Such need occurs, for example, in cardiacfailure and angina. Angina refers to a temporary chest pain that iscaused by insufficient blood reaching the heart, i.e. myocardialischemnia. The method is particularly useful if the mammal also requiresrelief of pain.

Accordingly, the present invention also provides a method for treatingcardiac failure or angina in a mammal in need thereof. The methodcomprises administering an effective amount of a selective mu-opioidreceptor agonist peptide described above to the mammal. The peptide ispreferably selected from the group consisting of Tyr-D-Agr-Phe-Lys-NH₂,2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Tyr-D-Ala-Phe-Phe,2′,6′-dimethyl-Tyr-D-Ala-Phe-Phe-NH₂ and2′,6′-dimethyl-Tyr-D-Arg-Phe-Orn-NH₂. Any mode of administration may beemployed. Systemic, oral or intrathecal administration is preferred.

The peptides of the invention are also useful before, during, and aftercardiac transplantation. During cardiac transplantation, a heart isremoved from a donor and is transplanted into a recipient.

The cardiac performance of the heart being transplanted is improved bytreating either or both of the donor and recipient in vivo with aneffective amount of a peptide of the invention. Tyr-D-Arg-Phe-Lys-NH₂,2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Tyr-D-Ala-Phe-Phe,2′,6′-dimethyl-Tyr-D-Ala-Phe-Phe-NH₂ and2′,6′-dimethyl-Tyr-D-Arg-Phe-Orn-NH₂ are preferred.

In addition, maintenance of the heart is improved after it has beenremoved from the donor, and before it is transferred to the recipient,by treating the heart ex vivo with a solution of the same peptidesdescribed above for iii vivo treatment.

The peptides of the present invention are also particularly useful fortreating pain before, during and after orthopedic surgery, includingjoint replacement surgery (hip, knee, shoulder, etc.). After orthopedicsurgery, patients are encouraged to begin rehabilitation as quickly aspossible. The sedative effects on motor function associated with opioidsof the prior art make immediate physical rehabilitation after surgicalprocedures difficult.

The peptides described above do not affect motor function as drasticallyas the opioids of the prior art. Therefore, the peptides of the presentinvention permit earlier physical rehabilitation of the patient.

A further advantage of the present invention is that the peptides do notcause uterine contraction. Controlling the rate and intensity of uterinecontractions during labor and delivery is an important aspect ofobstetric medicine. It is undesirable to produce unwanted uterinecontractions during pregnancy. Therefore, the claimed method is usefulfor relieving pain in pregnant women, including during labor anddelivery.

In addition, it has surprisingly been discovered that the peptides ofthe invention inhibit norepinephrine uptake. Norepinephrine inhibitionoccurs particularly in spinal cord synaptasomes when the peptides areadministered intrathecally

Accordingly, in a final embodiment, the invention relates to a methodfor inhibiting norepinephrine uptake. The method comprises administeringan effective amount of a peptide of the present invention to a mammal inneed thereof. Preferably the peptide is Tyr-D-Arg-Phe-Lys-NH₂,2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂, Tyr-DAla-Phe-Phe,2′,6′-dimethyl-Tyr-D-Ala-Phe-Phe-NH₂ and2′,6′-dimethyl-Tyr-D-ArgPhe-Orn-NH₂. The peptides can be administeredvia any mode of administration, including systemic, oral and intrathecaladministration.

An effective amount of the peptide will vary with the group of patients(age, sex, weight, etc.), the nature and severity of the condition to betreated, the particular polypeptide administered, and its route ofadministration, as is well known in the art. See the examples below foramounts administered to animals. Amounts suitable for administration tohumans are routinely determined by physicians and clinicians duringclinical trials.

While not being bound by theory or any particular mechanism, applicantsbelieve that the extraordinary analgesic potency of the peptides of thepresent invention may be due to the combination of mu-opioid agonism andenhancement of endogenous extraneuronal norepinephrine levels resultingfrom the inhibition of norepinephrine uptake.

Any dosage form known in the art of pharmacy is suitable. For oraladministration, liquid or solid dosage forms may be used. Some examplesof dosage forms include tablets, gelatin capsules, pills, troches,elixirs, suspensions, syrups, wafers, chewing gum and the like. Thepeptides can be mixed with a suitable pharmaceutical carrier (vehicle)or excipient as understood by practitioners in the art. Examples ofcarriers and excipients include starch, milk, sugar, certain types ofclay, gelatin, stearic acid or salts thereof, magnesium or calciumstearate, talc, vegetable fats or oils, gums and glycols.

For systemic or intrathecal administration, formulations of the peptidesmay utilize conventional diluents, carriers, etc., such as are known inthe art can be employed to deliver the peptides. The formulations maycomprise one or more of the following: a stabilizer, a surfactant,preferably a nonionic surfactant, and optionally a salt and/or abuffering agent. The peptide may be delivered in the form of an aqueoussolution, or in a lyophilized form.

The stabilizer may, for example, be an amino acid, such as for instance,glycine; or an oligosaccharide, such as for example, sucrose, tetralose,lactose or a dextram. Alternatively, the stabilizer may be a sugaralcohol, such as for instance, mannitol; or a combination thereof.Preferably the stabilizer or combination of stabilizers constitutes fromabout 0.1% to about 10% weight for weight of the proneurotrophin.

The surfactant is preferably a nonionic surfactant, such as apolysorbate. Some examples of suitable surfactants include Tween20,Tween80; a polyethylene glycol or a polyoxyethylene polyoxypropyleneglycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10%(w/v).

The salt or buffering agent may be any salt or buffering agent, such asfor example, sodium chloride, or sodium/potassium phosphate,respectively. Preferably, the buffering agent maintains the pH of thepharmaceutical composition in the range of about 5.5 to about 7.5. Thesalt and/or buffering agent is also useful to maintain the osmolality ata level suitable for administration to a human or an animal. Preferablythe salt or buffering agent is present at a roughly isotonicconcentration of about 150 mM to about 300 mM.

The formulations of the peptides of the present invention mayadditionally contain one or more conventional additive. Some examples ofsuch additives include a solubilizer such as for example, glycerol; anantioxidant such as for example, benzalkonium chloride (a mixture ofquaternary ammonium compounds, known as “quats”), benzyl alcohol,chloretone or chlorobutanol; anaesthetic agent such as for example amorphine derivative; or an isotonic agent etc., such as described above.As a further precaution against oxidation or other spoilage, thepharmaceutical compositions may be stored under nitrogen gas in vialssealed with impermeable stoppers.

The peptide may be administered via rapid intravenous bolus injection.Preferably, however, the peptide is administered as a constant rateintravenous infusion. The peptides of the invention may be administeredto mammals by sustained release, as is known in the art. Sustainedrelease administration is a method of drug delivery to achieve a certainlevel of the drug over a particular period of time. The level typicallyis measured by serum concentration.

The mammal can be any mammal, including, for example, farm animals, suchas sheep, pigs, cows, and horses; pet animals, such as dogs and cats;laboratory animals, such as rats, mice and rabbits, and humans. In viewof the limited ability of the peptides to cross the placental barrier,the method is especially effective for pregnant females, especiallypregnant women.

EXAMPLES Example 1 Experimental Methods for Determining AnalgesicPotency and Respiratory Effects of DALDA and [Dmt¹]DALDA AfterIntrathecal Administration in Rats

Animals

Male Sprague-Dawley Rats (300-350 g) were used.

Drugs and Drug Administration

[Dmt¹]DALDA and DALDA are synthesized by methods known in the art. See,for example, Brown, et al, U.S. Pat. No. 5,602,100 and Schiller et al,J. Med. Chem, 32, 698703 (1989). Naloxone hydrochloride was obtainedfrom Sigma, St. Louis, Mo. Each drug was dissolved in saline.Intrathecal delivery was performed either by direct injection or via anintrathecal catheter depending on the study.

1) Direct Percutaneous Injection.

Under light halothane anesthesia a needle connected to a Hamiltonsyringe was inserted percutaneously between spinal processes of thethird and fourth lumbar vertebrae into the intrathecal space. A quickflick of the tail was observed when the tip of the needle entered theintrathecal space and was used as an indicator of successful puncture(Mestre et al., 1994). Drugs were delivered in a volume of 5 μl.

2) Via Intrathecal Catheter

Intrathecal catheterization was performed at least two days prior to theexperiment as previously described (Shiinoyama, N. et al., 1996).Briefly, under halothane anesthesia, a PE-10 tube was inserted through asmall hole made in the atlanto-occipital membrane and threaded 8.5 cmdown the intrathecal space to the lumbosacral level of the spinal cord.Drugs were delivered via the catheter in a volume of 5 μl, followed by10 μl saline to flush the catheter.

Example 2

Analgesic Testing

To assess the antinociceptive effects of the opioids, the tail-flicktest was used. Radiant heat was applied to the tail at 5-8 cm from thetip using a tail-flick apparatus (IITC, Woodland Hills, Calif.). Thetime from the onset of the heat to the withdrawal of the tail(tail-flick latency) was measured. The intensity of the radiant heat wasadjusted so that baseline latencies would fall between 2.5 and 3.5seconds. To avoid tissue damage the heat stimulus was discontinued afterseven seconds. A baseline latency was obtained for each animal prior tothe administration of any drug. Subsequent response latencies weredetermined at designated time points. Analgesic testing was performed bya blinded investigator.

Study 1. Analgesic Potencies of Intratliecal Morphine (“MOR”), DALDA and[Dmt¹]DALDA

Cumulative dose-response studies were performed for each drug using thetail-flick test (Shimoyama, N. et al., 1996, Shimoyama, N. et al.,1997). Each drug was tested in a group of ten rats. After measuring thebaseline latencies, increasing doses of each drug were administered viaan intrathecal catheter until each animal in the group became ananalgesic responder. An analgesic responder was defined as one whoseresponse tail-flick latency was two or more times the value of thebaseline latency. The response latency after each dosing was determinedat peal analgesia, which was 15, 30 and 45 min after the administrationof the MOR, DALDA and [Dmt¹]DALDA, respectively (based on preliminarystudies). Any subsequent dosing was performed immediately after thedetermination of response latency. The percentage of analgesicresponders in the group of rats for each cumulative dose was calculated,and a cumulative dose-response curve was constructed. The dose-responsedata were analyzed by the BLISS-21 computer program. This programmaximized the log-likelihood function to fit a parallel set of gaussiannormal sigmoid curves to the dose-response data and provides ED₅₀values, 95% confidence interval (CI) and relative potency estimates(Umans and Inturrisi, 1981).

MOR, DALDA, and [Dmt¹]DALDA each produced a dose-dependentantinociceptive effect in the tail-flick test (FIG. 1). ED₅₀ values andpotency ratios obtained from the quantal dose-response curves are shownin Table 1. DALDA was 14 times more potent than MOR, whereas [Dmt¹]DALDAshowed a 3000-fold greater potency compared with MOR.

TABLE 1 ED₅₀ values and relative potencies of the antinociceptiveeffects of intrathecal MOR, DALDA, and [Dmt¹]DALDA in the rat tail-flicktest EDT₅₀ Compound pmol 95% CI Potency Ratios Morphine 3330 1940-5600 1DALDA 237 149-370 14.1 [Dmt¹]DALDA 1.06 0.64-1.71 3160Study 2. Naloxone Reversal of Antinociceptive Effects

The effect of naloxone on the antinociceptive effects of intrathecalMOR, DALDA and [Dmt¹]DALDA was examined. An equipotent dose (3 times theED₅₀ value determined in Study 1) of MOR, DALDA or [Dmt¹]DALDA was givenvia an intrathecal catheter. Tail-flick latencies were measured prior tothe administration of any drug and at the time of peak analgesia foreach compound (at 15, 30 and 45 minutes after administration, for MOR,DALDA and [Dmt¹]DALDA, respectively). Naloxone hydrochloride at a doseof 82.5 nmol or saline was administered via the intrathecal catheter 10minutes prior to the second tail-flick testing. Four rats were testedfor each combination of drugs. Data were analyzed using the pairedt-test.

In rats that received MOR, DALDA, or [Dmt¹]DALDA followed by saline, alltail-flick latencies reached cut-off (7 s) at the time of peak effect ofeach agonist. When naloxone (82.5 mnol) was administered instead ofsaline, the tail-flick latencies measured at the time of peak effectwere not different from baseline values (Table 2).

A dose 3 times the ED₅₀ value of MOR, DALDA, or [Dmt¹]DALD)A wasadministered intrathecally. Tail-flick latencies were measured prior tothe administration of any drug (=baseline latency) and at the time ofpeak analgesia (=response latency). Nalaxone hydrochloride at 82.5 nmolor saline was administered intrathecally 10 min prior to the secondtail-flick measurement (n=4 for each group). The results are shown inTable 2.

TABLE 2 Reversal of antinociceptive effects of MOR, DALDA, and[Dmt¹]DALDA by nalozone Compounds Baseline Latencies Response LatenciesMOR + saline 2.78 ± 0.50 7 (cut-off latency)* DALDA + saline 2.63 ± 0.157 (cut-off latency)* [Dmt¹]DALDA + saline 2.83 ± 0.17 7 (cut-offlatency)* MOR + naloxone 3.08 ± 0.31 3.13 ± 0.41 DALDA + naloxone 2.65 ±0.17 2.90 ± 0.43 [Dmt¹]DALDA + naloxone 2.88 ± 0.10 2.68 ± 0.15*Significantly different tram baseline (p < 0.05).Study 3. Time Course of Antinociceptive Effects

Equipotent doses (3 times the ED₅₀ value obtained in Study 1) of MOR,DALDA and [Dmt¹]DALDA were given intrathecally by direct percutaneousinjection. Tail-flick latencies were measured prior to and every hour upto 15 hours after the administration of each drug. The number of ratstested for each drug was 8. Data were analyzed using the one-wayanalysis of variance followed by the Dunnett's test.

As shown in FIG. 2, MOR, DALDA, and [Dmt¹]DALDA showed different timecourses of antinociceptive effects after intrathecal administration ofequipotent doses of the three drugs (3 times the ED₅₀ value forantinociceptive effect). The tail-flick latencies were significantlygreater than baseline for 3, 7, and 13 h after the intrathecaladministration of MOR, DALDA, and [Dmt¹]DALDA, respectively. Alltail-flick latencies returned to baseline by the end of the experiment.

Study 4. Respiratory Effects of Intiathecal MOR, DALDA and [Dmt¹]DALDA

The effects of each drug on minute ventilation (VE) under 5% CO₂challenge were evaluated using whole body plethysmography (Tatsumi etal., 1991). An unrestrained rat was placed in a 3-liter whole-bodyplethysmograph chamber. After a 15 min acclimation period, a gas mixtureof 5% CO₂ and 21% O₂ in N₂ (100% humidified) was supplied into and outof the chamber at a rate of 1000 mi/min, and the animal was allowed tobreathe the gas mixture for 5 minutes. After a steady-state ventilatorycondition had been reached, with the animal awake and quiet, the inletand outlet of the chamber were closed and the pressure changes in thebox (due to the warming and wetting of the gas inspired by the rat andthe cooling and drying of the expired gas) were recorded using ahigh-gain differential pressure transducer. A calibration volume of 0.2ml of air was regularly introduced into the chamber during therecordings. The recordings were made for 20 to 30 seconds. Tidal volumeswere calculated from the pressure changes using the equation derived byDrorbaugh and Fenn (1955). Respiratory frequencies were determined fromthe number of respiratory cycles in the recordings and VE values werecalculated (tidal volume×frequency).

Rats were randomly assigned to one of 7 groups. The number of rats ineach group was 6 to 10. The animals of each group were given 3 times(lower dose) or 30 times (higher dose) the ED₅₀ value (antinociceptive)of MOR, DALDA or [Dmt¹]DALDA, or saline by direct percutaneousintrathecal injection. VE under 5% CO₂ challenge was determined prior toand every hour up to 10 hours after the administration of the drug. VEvalues were expressed as a percentage of the baseline VE value obtainedprior to the administration of any drug. The smallest VE value obtainedafter the administration of a drug (minimum V E) was determined for eachanimal. Mean values of the minimum V E for animals of each group werecompared using the one-way analysis of variance followed by theDunnett's test. For each animal, respiratory depression was defined as aminimum VE value that is below two standard deviations of the mean(mean−2×SD) of the saline group.

The effects of MOR, DALDA, and [Dmt¹]DALDA on minimum VE are illustratedin FIG. 3. Compared with the group that received saline, the minimum VEwas significantly lower in the group that received the high dose of MOR(30 times the antinociceptive ED₅₀) but not in the group that receivedthe low dose of MOR (3 times the antinociceptive ED₅₀). Both the low-and high-dose DALDA groups showed a significantly lower minimum VE. Incontrast, the minimum VE was not different in the low- or high-dose of[Dmt¹]DALDA. When the minimum VE of each animal was checked to determinewhether it satisfied the criterion set for respiratory depression [lessthan a critical value of mean—(2×S.D.) of the minimum VE of the salinegroup], a substantial number of animals in the groups that showed adecrease in the mean value of minimum VE had a minimum VE value lowerthan the critical level (a “low minimum VE”) (Table 4). Furthermore, oneanimal of the lower dose MOR group and one animal of the higher dose.[Dmt¹]DALDA group also had a low minimum VE, although the mean values ofthe groups were not significantly different from the saline group. Noanimal in the lower dose [Dmt¹]DALDA group showed a low minimum VE. Thetiming of the occurrence of low minimum VE for each animal was between 3and 5 h after administration for all groups.

Doses of 3 times the ED₅₀ value and 30 times the ED₅₀ value(antinotioeptive) of each drug were administered. A minimum VE value(see Materials and Methods) less than the mean—(2×S. D.) of the minimumVE value of the saline group was considered a “low minimum VE”. Theresults are shown in Table 3.

TABLE 3 The occurrence of respiratory depression after intrathecaladministration of MOR, DALDA, and [Dmt¹]DALDA Total No. No. of Rats withTime of Occurrence of Drug Dose of Rats Low Minimum VE Low Minimum VESaline 7 0 Morphine 10 nmol 6 1 4 h after administration 100 nmol 10 43-5 h after administration DALDA 0.7 nmol 6 4 3-5 h after administration7 nmol 8 4 3-5 h after administration [Dmt¹]DALDA 3.4 pmol 6 0 34 pmol 71 3 h after administrationStudy 5: Effects of α₂-Adrenergic Blockade.

Effect of α₂-Adrenergic Blockade on Antinociceptive Action of[Dmt¹]DALDA and DALDA. The antinociceptive effect of intrathecal[DMT¹]DALDA and DALDA were compared in the absence and presence ofintrathecal yohimbine, an α₂-adrenergic antagonist. Rats wereadministered [DMT¹]DALDA alone (1.1 pmol, n=10), [DMT¹]DALDA (1.1 pmol)and yohimbine (100 μg) (n=10), DALDA alone (240 pmol, n=9), DALDA (240pmol) and yohimbine (100 μg) (n=9), or yohimbine alone (100 μg) (n=6).Due to the limited solubility of yohimbine, the drug solutions wereprepared with 50% dimethyl sulfoxide in saline and delivered in a volumeof 104 l. Intrathecal administration of 10 μL of 50% dimethyl sulfoxidein saline by itself did not have any effect on tail-flick latency (n=4,data not shown). Tail-flick latencies were measured before and every 20min up to 120 min after drug administration. Within-group differenceswere analyzed by one-way ANOVA and between-group differences wereanalyzed by two-way ANOVA.

Yohimbine (100 μg) alone had no effect on tail-flick latency at any timecompared with baseline value (data not shown). The addition of yohimbine(100 μg) significantly attenuated the antinociceptive effect of[Dmt¹]DALDA (1.1 pmol) (FIG. 4), but not DALDA (240 pmol) (FIG. 5).

Behavioral Effects: Rigidity of the caudal part of the body was observedin each animal that received the higher dose (see study 3) of MOR,DALDA, or [Dmt¹]DALDA. Four rats in the group that was given the higherdose of DALDA and had a minimum VE less than 60% of baseline (FIG. 3)showed sedative effects that coincided with the period of respiratorydepression. During this period, normal activity was markedly suppressedin these animals and they could not negotiate a 60° mesh (Shimoyama et.Al., 1997). However, they retained their righting reflex. No overtsedative effects were observed in animals of other groups.Inhibition of NE and 5-HT Uptake in Spinal Cord Synaptosomes. DALDA,[Dmt¹]DALDA, and MOR all inhibited [³H]NE uptake in a dose-dependentmanner (FIG. 6). The IC₅₀ for inhibition of [³H]NE uptake was 4.1 μM(2.5-6.7) for [Dmt¹]DALDA, 54 μM (28-107) for DALDA, and 870 μM(197-3832) for MOR. At a dose of 10⁻⁴ [Dmt¹]DALDA inhibited [³H]NEuptake by 80.6±1.5%. Neither DALDA nor [Dmt¹]DALDA had any effect on5-HT uptake in spinal cord synaptosomes (data not shown).

Example 3 Experimental Methods for Determining Analgesic Potency ofSubcutaneous [Dmt¹]DALDA in Mice

Antinociceptive Assay.

The antinociceptive assay was a modification of the radiant tail-flicktest described by Tulunay and Takemori (1974). The data were madequantal by designating apositive antinociceptive response as oneexhibiting an increased latency to tail-flick of at least 3 SD above themean latency of animals not given the drug.

Intrathecal Injections.

Drugs were injected i.t. (Hylden and Wilcox, 1980) in a volume of 5ul/mouse, and the latencies measured 30 min after the injections.

Tolerance Measurement.

Mice were made tolerant to morphine by s.c. implantation of one morphinepellet (containing 75 mg morphine free base) for 72 hr. The degree oftolerance was determined as the ratio of the AD50 value of agonist inmorphine-pelletted mice to that of placebo-pelletted mice (Way et al.,1969). The implanted morphine pellet was left intact during theantinociceptive assay.

Example 4 Experimental Methods for Determining in Vitro and in VivoPharmacokinetics of DALDA and [Dmt¹]DALDA in Sheep

Animal Preparation Chronic indwelling catheters were surgically placedin the descending aorta and inferior vena cava of adult female sheepunder epidural anesthesia. Five or more days were allowed for recoveryfrom surgery prior to experimental studies.

Compounds [Dmt¹]DALDA was prepared by solid-phase synthesis as describedpreviously (Schiller et al., 2000). For the synthesis of the deuteratedpeptide analogs, Boc-Phe(d₅)—OH was used in place of Boc-Phe-OH.Pentadeuterophenylalanine was purchased from C/D/N Isotope, Vaudreuil,Quebec, Canada and was converted to Boc-Phe(d₅)—OH by reacting withdi-tert-butyldicarbonate. The deuterated peptides were purified bysemi-preparative reversed-phase HPLC (Schiller et al., 1989). The purityof all peptides was verified by fast atom bombardment-mass spectrometry(FAB-MS), and the correct amino acid sequence was confirmed by tandemmass spectrometry (Schiller et al., 1989).In vitro degradation studies To study the degradation of the threepeptide analogs in blood, DAMGO (50 μg), DALDA (10 μg) or [Dmt¹]DALDA(10 μg) was added to 25 ml of freshly collected sheep blood, and themixture was incubated in a water bath at 37° C. Three ml of blood wasremoved from the blood sample at 15 s and at 5, 15, 30, 60 and 120 minafter peptide addition. The blood sample was gently mixed throughout theentire incubation period.In vivo pharmacokinetic studies In order to avoid the rapid distributionphase associated with intravenous bolus administration, all threepeptides were administered as constant rate intravenous infusions tosheep. Based on the known elimination half-life of ˜1.5 hr for DALDA(Szeto et al., 1998), it was estimated that a 4-hr infusion would allowplasma drug levels to approach steady state levels. DALDA (0.6 mg/kg/hr)and [Dmt¹]DALDA (0.06 mg/kg/hr) were, therefore, infused via the venouscatheter for a period of4 hr. [Dmt¹]DALDA was infused at a lower dosebecause it was found to be 200-fold more potent than DALDA afterintrathecal administration in the rat tail flick test (Shimoyama et al.,submitted for publication) and 100-fold more potent than DALDA inincreasing blood pressure in sheep (unpublished data). The current limitof sensitivity of the analytical method prevented the use of an evenlower dose of [Dmt¹]DALDA. Blood samples (5 ml) were collected from thearterial catheter at 0, 1, 2, 3, 3.5, 4, 4.25, 4.5, 5, 6 and 7 hr.Because preliminary data showed much more rapid elimination of DAMGO insheep, DAMGO (0.6 mg/kg/hr) was only infused for 3 h, and blood sampleswere collected at 0, 0.5, 1, 2, 2.5, 3, 3.25, 3.5, 3.75, 4 and 4.5 hr.Quantitative analysis of DAMGO, DALDA and [Dmt¹]DALDA All blood sampleswere collected into chilled borosilicate glass tubes that containedEDTA, and were centrifuged; the plasma was stored in glass containerswith teflon-lined caps, and was frozen at −80° C. until processed. Allthree peptides were purified by HPLC and quantified with massspectrometry. Details of the quantitative method for DALDA and DAMGOhave been published (Grigoriants et al., 1997; Desiderio et al., 2000)and will only be presented briefly here. All plasma samples weredeproteinated and eluted through a solid phase extraction cartridge(Sep-Palc C18, Millipore Corp., Milford, Mass.) with CH₃CN. An internalstandard, the respective deuterated peptide analog(H-Tyr-DAla-Gly-MePhe(d₅)-Gly-ol, H-Tyr-D-Arg-Phe(d₅)-Lys-NH₂, orH-Dmt-D-Arg-Phe(d₅)Lys-NH₂), was added to each plasma sample beforedeproteinization. The filtered plasma sample was chromatographed on anRP-analytical column (Delta Pak, 5 μ, C18, 150×3.9 mm, Waters, Milford,Mass.) at a flow rate of 1.5 ml mind⁻¹, and UV absorption was monitoredat 200 nm (Varian Assoc. Inc., Walnut Creek, Calif.). Gradient elution(7→30% acetonitrile in 0.1% trifluoroacetic acid; 30 min) was used.One-minute fractions were collected, and each fraction was lyophilizedfor MS analysis. A matrix-assisted laser desorption/ionization (NALDI)time-of-flight (TOF) mass spectrometer (Voyager™-DE RP BiospectrometryWorkstation, PerSeptive Biosystems Inc, Framingham, Mass.) was used toquantify the peptide in each plasma sample. The (M+H)+ ion current foreach peptide was compared to the ion current from the d₅-peptide. (M+H)⁺data were used to quantify DALDA and [Dmt¹]DALDA; no intense signal thatincluded the d5 label was available. A 9:1 signal-to-noise (S/N) ratiowas found for DALDA and [Dmt¹]DALDA at a concentration of 375 fmol/μl.Recalculating to a 3:1 S/N ratio, a limit of detection of 125 fmol/μl isavailable. A post-source decay (PID) fragment was available for DAMGO.An S/N ratio of 10:1 was measured at 4 pmol/μl. Thus, a calculated limitof detection of 400 fmol/μl is availablePharmacokinetic Analyses Plasma levels of DAMGO, DALDA and [Dmt¹]DALDAduring and after the 4 h infusion were subjected to compartmentalanalysis using nonlinear regression (WINNONLIN). The derivedpharmacokinetic constants were used to calculate the apparent volume ofdistribution at steady state (Vd), elimination half-life (t_(1/2)) andclearance (CL). Example 5

Experimental Methods for Determining Whether DALDA and [Dmt¹]DALDA areSubstrates for the Peptide Transporter PEPT2.

Rat PEPT2 is expressed in HeLa cells heterologously by using thevaccinia virus expression technique. HeLa cells do not express anypeptide transport system endogenously. The activity of theheterologously expressed PEPT2 in these cells is measured by monitoringthe uptake of the model dipeptide glycylsarcosine (50 micromolar) in thepresence of an inwardly directed proton gradient. The proton gradient isthe driving force for the peptide transporter. Therefore, the uptakebuffer used in these studies is 25 mnM Mes/Tris (pH 6.0) containing 140mM NaCl, 5.4 mM KC1, 0.8 mM MgSO4, 1.8 mM CaC12 and 5 mM glucose. HeLacells that were transfected with empty vector (pSPORT) serve ascontrols. The uptake of glycylsarcosine in these control cells issubtracted from the uptake in ratPEPT2 cDNA-transfected cells tocalculate the uptake that is mediated specifically by rat PEPT2. Weperformed a dose-response experiment for DALDA and superDALDA over aconcentration range of 10-1000 micromolar. The data from thesedose-response experiments were used to calculate the IC50 values forthese two compounds.

Example 6 Description of Studies Showing [Dmt¹]DALDA does not IncreaseBlood Pressure if Administered as a Constant Rate Intravenous Infusion.

Earlier studies showed that [Dmt¹]DALDA produces a transient increase inblood pressure in awake sheep when administered as an intravenous bolusat doses ranging from 0.003-0.009 mg/kg. Even a brief period ofincreased blood pressure may be a risk factor in patients withhypertension, compromised myocardial function (such as after amyocardial infarction) or patients who are at risk for strokes. Theresponse is short-lasting because the receptor desensitizes rapidly. Wecan take advantage of this rapid desensitization and eliminate the bloodpressure response by administering the drug as a slow intravenousinfusion rather than as a rapid bolus injection.

Studies have been carried out in sheep whereby [Dmt¹]DALDA was infusedintravenously at a constant rate ranging from 0.06-0.6 mg/kg/h for aslong as 4 h. There was no significant change in blood pressure observedat any time during or after the drug infusion. Thus the increase inblood pressure can be avoided by using a slow intravenous infusion of[Dmt¹]DALDA.

Example 7 Experimental methods for determining effects of DALDA and[Dmtl]DALDA in Inhibiting Norepinephrine Uptake in Cardiac and SpinalCord Synaptosomes

Preparation of Heart and Spinal Cord Synaptosome

Adult female Hartley strain guinea pigs (440-550 g) were decapitated andthe spinal cords taken out. A crude synaptosomal (P2) fraction wasprepared as described previously (Lonart and Johnson 1995; Li 2000).Briefly, the tissue was minced and homogenized in 10 ml of ice-cold 0.32M sucrose solution (pH 7.4) with a Thomas B075 homogenizor, clearance0.13-0.18 mm. The homogenate was centrifuged for 10 min at 1,200×g in aMegafuge centrifuge at 4° C. The resulting supernatant was thencentrifuged at 20,000 ×g for 20 min in JA-17 rotor of a Beckman J2-21centrifuge at 4° C. and the supernatant discarded. The pellet (P2) wasgently resuspended in aerated (100% O₂) ice-cold buffer contairing (inmM):HEPES 20, NaCl 140, KCl 5, NaHCO₃ 5, MgCl₂ 1, Na₂HPO₄ 1.2, CaCl₂ 1.2and glucose 10, pH 7.4, and centrifuge at 3,000×g for 12 min in aMegafuge at 4° C. Pour out the supernatant. Gently resuspend synaptosomein 10 ml buffer with the aid of a Dotince homogenizor just before using.Protein concentration was determined by the method of Bradford (1976) at595 nm using bovine serum albumin as a standard in a Spectra Max plusmachine (Molecular Device Co. Sunnyvale, Calif.)

Uptake Experiments

Add buffer 0.6 ml in tubes, synaptosome solution 0.2 ml (about 100 μg),then varying concentration of drug 0.1 ml (each concentration performedin triplicate), put in water bath for pre-incubation for 10 min at 37°C. The uptake was initiated by the addition of [³H]NE (100 nM) or[³H]5-HT (50 nM) 0.1 ml, continued to incubate for 6 min and terminatedby a rapid cooling of sample tubes in ice-cold water for 3 min. Thesynaptosome were then collected using a Harvest machine with GF/Bfilter, washed for 3 times with ice-cold 150 mM Tris HCl buffer (pH 7.4)2 ml. After drying, put filter in scintillation liquid vials then addscintillation liquid 6 ml. Filter-bound radioactivity was counted byliquid scintillation counter (Becklman LS6001). The difference in [³H]NEor [³H]5-HT accumulation at 37° C. and 0° C. was taken as a measure ofactive uptake.

Data Analysis

Results represent with mean values±SEM of at least three to fiveindependent experiments. The IC₅₀ were calculated by GraphPad Prismprogram and also represent with 95% confidence intervals.

Drugs

1-[7,8-³H]Norepinephrine (specific activity 1.37TBq/mmol, radiochemicalpurity 93.2%), and 5-hydroxy[³H]trytamine trifluoroacetate (specificactivity 4.00TBq/mmol, radiochemical purity 99%) were purchased fromAmersham Phannacia Biotech Co. (Buckingharnshire, England). Desipraminehydrochloride, fluoxetine hydrochloride and morphine sulfate were boughtfrom Sigmra Co. (St. Louis, Mo.). DynorphinA(1-13), DAMGO were obtainedfrom NIH. [Dmt¹]DALDA and DALDA are synthesized by methods known in theart. See, for example, Brown, et al, U.S. Pat. No. 5,602,100 and theexample below.

Example 8 Peptide Synthesis

Peptides are synthesized by the solid-phase method usingtert-butyloxycarbonyl (Boc)-protected amino acids and1,3-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) ascoupling agents. Boc-Tmt is prepared by methods kcnown in the art.2′-methylphenylalanine can be prepared from1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) with minimalracemization (8%) by carrying out the hydrogenolysis at 60° C. using 10%Pd/C and 4 atm H₂ pressure. Under the latter conditions,2′-methyltyrosine (Mmt) is prepared from Tic(OH) in good yield (48%) andwith minimal racemization (<10%). Racemic 2′-hydroxy-6′-methyltyrosineis synthesized by methods known in the art. The bis-Boc derivative ofMmt and the tris-Boc derivative of D,L-Hmt are prepared by reaction withdi-tert-butyl dicarbonate in the presence of triethylamine (TEA) and4-dimethylaminopyridine (DMAP). The tris-Boc derivative of D,L-Hmt isincorporated into the peptide in racemic form. Peptides are cleaved fromthe resin by HF/anisole treatment in the usual manner. Peptidepurification and separation of the diasteromeric peptides in the case ofthe Hmt¹-analogue were achieved by preparative reversed phase HPLC.

1. A method for protecting a mammal's heart from ischemic-reperfusioninjury in a mammal in need thereof, the method comprising administeringto the mammal an effective amount of2′,6′-dimethyl-Tyr-D-Arg-Phe-Lys-NH₂ peptide; wherein said effectiveamount is effective to protect the mammal's heart from ischemicreperfusion injury.
 2. The method according to claim 1, wherein themammal is a human.
 3. The method according to claim 1, wherein thepeptide is administered via constant rate intravenous infusion.
 4. Themethod according to claim 1, wherein said peptide is mixed with asuitable pharmaceutical carrier.